The overarching goal of this proposal is to investigate the Hippo signaling pathway in the liver and how it contributes to the pathogenesis of hepatic fibrosis. The outcome of these studies will impact on our understanding of hepatic development and recovery after injury. The Hippo signaling pathway is an important regulator of liver size, cellular proliferation and cell fate. The Hippo pathway negatively regulates Yap. High levels of Yap in hepatocytes convert these cells into a hepatic progenitor cell (HPC)-like phenotype. Rapidly dividing HPCs are associated with the development of fibrosis, and it is known that Yap directly activates genes associated with this process. Cirrhotic liver samples often display nuclear Yap staining (a sign of Hippo inactivation) and hepatic stellate cell (HSC) activation is temporally associated with increasing Yap. These observations suggest that inactivating Hippo signaling in epithelial cells can activate a profibrotic gene program. This proposal will validate and dissect the mechanism of this observation. In AIM 1, several fibrogenic liver injury models will be examined to see if Hippo signaling is inactivated. This will be complemented with loss of function studies in the epithelial compartment to determine if abrogating Hippo signaling will minimize the development of fibrosis. In AIM 2, Yap-expressing hepatocytes will be examined by cellular fractionation and using single-cell sequencing techniques to determine if a priori, a particular hepatocyte type is most sensitive to Yap expression and if this cell-type likely orchestrates the development of liver fibrosis. AIM 3, focuses on determining which secreted factors from Yap-expressing hepatocytes likely activate HSCs. This will be performed using RNA interference and Cas9-Crispr technologies to knockdown expression of candidate molecules defined by microarray expression and ChIP-Seq data from Yap overexpressing hepatocytes. This screen will be performed in vitro followed by in vivo validation. Understanding if Hippo signaling is a common mechanism for the development of hepatic fibrosis, the characteristics of the epithelial cells that may orchestrate this process, and potential secreted factors to activate HSCs will be important information in developing therapeutics to treat liver fibrosis.